Alveolar epithelial cell keratin expression during lung development.

Defining the expression and organization of keratins has provided insight into epithelial cell differentiation during tissue development and remodeling. We have used monoclonal antibodies to examine keratin distribution in lung epithelial cells in the rat from the preglandular phase of gestation to the adult. Of particular interest were the distributions of keratin No. 18 and keratin No. 19, since previous results have suggested these keratins may be important in alveolar epithelial cell transitions occurring in adult remodeling lung and in cultured type II cells. The epithelial tubes at 15 days of gestation do not react with 24A3 monoclonal antibody to keratin No. 18, nor is this antigen apparent by gel or immunoblot analysis. Staining is apparent at day 16, however, showing a light punctate pattern at the basal edge of the cells, and becomes prominent by day 17, with intensity greatest in the larger airway tubes. The intensity and number of cells in the parenchyma staining with 24A3 peaks at postnatal days 5 to 10, when proliferation and cytodifferentiation of type I and type II cells is most active. In the adult, staining of type II cells is present mainly at the cell periphery, and occasional reactive attenuated type I-like cells can be observed. Keratin No. 19 immunoreactivity is not present in the primitive epithelial tube until 19 days' gestation but predominantly stains type II pneumocytes in the adult rat lung throughout the entire cell. AE3 antibody to basic keratins stains similarly to keratin No. 19. We conclude that keratin No. 18 is expressed at high levels in type II cells during development in periods of intense proliferation and alveolarization. This correlates with our previous observations on keratin expression following bleomycin lung injury.     

Regulation of 1-cys peroxiredoxin expression in lung epithelial cells

1-cys peroxiredoxin (1-cys Prx), the only member of a Prx subfamily that contains a single conserved cysteine residue, is abundant in lung. This bifunctional protein has both glutathione peroxidase and phospholipase A2 activities compatible with a role both in protection against lung oxidant injury and also in lung phospholipid metabolism. Here we studied the developmental expression of 1-cys Prx in rat lungs and hormonal effects on protein expression in human and rat lung cells. There was little change in 1-cys Prx expression during the prenatal period, but a marked increase in expression immediately after birth. Enzymatic (peroxidase and phospholipase) activities increased gradually after birth and reached adult level at 7-14 postnatal days. Expression of the protein was induced in the presence of dexamethasone (Dex) in cultured human and rat lung epithelial cells and also was upregulated in neonatal rat lung in vivo. cAMP treatment had no effect on expression, although there was a modest synergistic effect when combined with Dex in human fetal lung epithelial cells. The increased expression of 1-cys Prx at birth may be important for surfactant phospholipid turnover related to the phopholipase A2 activity of the protein and for antioxidant defense based on its peroxidase function.     

Spatiotemporal expression of zic genes during vertebrate inner ear development

BACKGROUND: Inner ear development involves signaling from surrounding tissues, including the adjacent hindbrain, periotic mesenchyme, and notochord. These signals include SHH, FGFs, BMPs, and WNTs from the hindbrain and SHH from the notochord. Zic genes, which are expressed in the dorsal neural tube and act during neural development, have been implicated as effectors of these pathways. This report examines whether Zic genes' involvement in inner ear development is a tenable hypothesis based on their expression patterns. RESULTS: In the developing inner ear of both the chick and mouse, all of the Zic genes were expressed in the dorsal neural tube and variably in the periotic mesenchyme, but expression of the Zic genes in the otic epithelium was not found. The onset of expression differed among the Zic genes; within any given region surrounding the otic epithelium, multiple Zic genes were expressed in the same place at the same time. CONCLUSIONS: Zic gene expression in the region of the developing inner ear is similar between mouse and chick. Zic expression domains overlap with sites of WNT and SHH signaling during otocyst patterning, suggesting a role for Zic genes in modulating signaling from these pathways. Developmental Dynamics 242:897–908, 2013. © 2013 Wiley Periodicals, Inc.     

Type II pulmonary epithelial cells of the newborn opossum lung

The air chambers of the newborn opossum are lined by a respiratory epithelium which appears similar to that lining alveoli of other species. The type II pulmonary epithelial cell is cuboidal in shape, shows apical microvilli, several Golgi complexes, lipid droplets, and numerous cytosomes. The cytosomes contain osmiophilic lamellae and are similar in appearance to whorls of osmiophilic material lying free within the alveolar lumina and on the surface of the respiratory lining epithelium. In the newborn opossum lung, examination of the air chambers with the scanning electron microscope permits the observation of several type II pulmonary epithelial cells in a single field. The potential use of the air chamber of the newborn opossum as an experimental model is suggested.     

Flk-1在早产儿视网膜病大鼠模型中的表达和作用

目的：探讨血管内皮生长因子受体-2（flk-1）的表达与早产儿视网膜病（ROP）视网膜血管变化的关系。 方法： 86例新生未成熟SD大鼠随机分为高氧组和对照组，各组再随机分为1、3、7及14 d 4小组。高氧组吸入75%的氧（7 d后置入空气中饲养）建立ROP大鼠模型，对照组则在空气中饲养。取视网膜组织制作标本，HE染色及CD34标记血管内皮细胞观察视网膜血管改变，采用免疫组织化学方法测定flk-1在视网膜的表达。 结果： ①随着天数的增加对照组毛细血管密度指数（RCDI）呈逐渐上升（P<0.01）,7 d时高氧组RCDI最低,停氧7 d(即14 d)时增加（P<0.01）；②7 d时flk-1在对照组视网膜中表达最强，与第1 d和第14 d比较，表达强度均具有显著差异（P<0.05）；高氧组7 d时与对照组比较， flk-1的表达明显减弱，统计学处理具有显著差异（P<0.05）；而高氧组14 d flk-1的表达增强，与对照组比较，差异显著（P<0.05）。 结论： 当未成熟的视网膜处于高氧状态时，flk-1表达减弱，视网膜血管减少；当视网膜处于相对低氧状态时， flk-1表达增强，视网膜血管出现了明显的增生。flk-1在视网膜血管发育和ROP的血管改变过程中起着重要作用。     

Kinetics of pulmonary epithelial proliferation during prenatal growth of the mouse lung

Autoradiographic methods were used to analyze cell kinetics in peripheral epithelium of the fetal mouse lung, days 15-18. The ³H labeling index decreased in proportion to increases in fetal lung and body weight from day 15 to day 18. Cell-cycle determination estimated from the percent of labeled metaphases at intervals after ³H thymidine injection showed the shortest cell-cycle time (12.25 hours) on day 15 during the precenal stage of growth. Total cycle time (Tc) increased by approximately one hour on each subsequent day. Between days 15 and 16 Tc increased due to a 1-hour increase in the post mitotic interphase (G₁); thereafter increases in Tc could be ascribed almost entirely to lengthening of the DNA synthetic period. A comparison of cell-cycle time in central and peripheral tubules of the same lungs on day 15 showed a slower cell cycle in the central tubules than in the peripheral branches. An estimate of the growth fraction (GF) on each day based on calculated and observed cell-cycle times showed that GF was 1 on days 15 and 16, decreasing to 0.69 on day 17 and 0.27 on day 18. Thus cell-cycle time increases with lung maturation and it increases from peripheral to central zones of the bronchial tree. The greater part of the observed decline in growth rate as the prenatal phase of growth is completed was due largely to a decrease in the fraction of cells in the cell cycle.     

Spatiotemporal dynamics of microRNA during epithelial collective cell migration

MicroRNAs (miRNAs) are small, noncoding RNAs variably involved in a wide variety of developmental and regenerative programs. Techniques for monitoring the spatiotemporal expression of miRNA in living cells are essential to elucidate the roles of miRNA during these complex regulatory processes. The small size, low abundance, sequence similarity, and degradation susceptibility of miRNAs, however, make their detection challenging. In this study, we detail a double-stranded locked nucleic acid (dsLNA) probe for detecting intracellular miRNAs during epithelial collective migration. The dsLNA probe is capable of detecting the dynamic regulation and dose-dependent modulation of miRNAs. The probe is applied to monitor the spatial distribution of miRNA expression of a migrating epithelium. Our results reveal a gradient of miRNA over the first one hundred microns from the leading edge and show the involvement of miR-21 in the complex regulation of transforming growth factor beta modulated epithelial migration. With its ease of use and capacity for real-time monitoring of miRNAs in living cells, the dsLNA probe carries the potential for studying the function and regulation of miRNA in a wide spectrum of complex biological processes.     

Kinetics of pulmonary epithelial proliferation during prenatal growth of the mouse lung.

Autoradiographic methods were used to analyze cell kinetics in peripheral epithelium of the fetal mouse lung, days 15-18. The 3H labeling index decreased in proportion to increases in fetal lung and body weight from day 15 to day 18. Cell-cycle determination estimated from the percent of labeled metaphases at intervals after 3H thymidine injection showed the shortest cell-cycle time (12.25 hours) on day 15 during the precanal stage of growth. Total cycle time (Tc) increased by approximately one hour on each subsequent day. Between days 15 and 16 Tc increased due to a 1-hour increase in the post mitotic interphase (G1); thereafter, increases in Tc could be ascribed almost entirely to lengthening of the DNA symthetic period. A comparison of cell-cycle time in central and peripheral tubules of the same lungs on day 15 showed a slower cell cycle in the central tubules than in the peripheral branches. An estimate of the growth fraction (GF) on each day based on calculated and ovserved cell-cycle time showed that GF was 1 on days 15 and 16, decreasing to 0.69 on day 17 and 0.27 on day 18. Thus cell-cycle time increases with lung maturation and it increases from peripheral to central zones of the bronchial tree. The greater part of the observed decline in growth rate as the prenatal phase of growth is completed was due largely to a decrease in the fraction of cells in the cell cycle.     

Interstitial exclusion of albumin in rabbit lung during development of pulmonary oedema

The modifications of the macromolecular sieving properties of the pulmonary extracellular tissue matrix were studied in adult anaesthetized rabbits ( n = 10) exposed to increased tissue hydration. Exclusion of albumin from the perivascular pulmonary interstitial space was determined by using the continuous infusion method coupled with direct sampling of interstitial fluid performed through the wick technique. The rabbits underwent an intravenous infusion of saline amounting to 10 ( n = 5) or 20 % ( n = 5) body weight. Extracellular albumin distribution volume was derived from the steady state tissue concentration of radioactive rabbit serum albumin (¹²⁵I-RSA). Pulmonary extracellular and intravascular fluid volumes ( V _x and V _v, respectively) were measured as distribution volumes of ⁵¹Cr-EDTA and ¹³¹I-RSA, respectively, and interstitial fluid tracer concentrations were determined in interstitial fluid collected through implanted wicks. At the highest degree of hydration, interstitial fluid volume ( V _i= V _x− V _v) and extravascular albumin distribution volume ( V _a,w) significantly increased by 38.5 and 240.2 %, respectively, compared to control. Albumin-excluded volume ( V _e,a= V _i− V _a,w) was 398.9 ± 17 μl (g dry tissue weight)⁻¹; the albumin-excluded volume fraction ( F _e,a= V _e,a/ V _i) was 0.23 ± 0.01, 33.2 % of the control value. Data indicate that, at variance with what is observed in tissues like skin and muscle, pulmonary F _e,a is highly sensitive to tissue fluid content.     

Spatiotemporal transcription of connexin45 during brain development results in neuronal expression in adult mice

Characterization of the expression pattern of connexins in neural tissue is a necessary prerequisite for understanding the functional relevance of the corresponding gap junction channels in brain. Here we describe the cell type-specific expression of connexin45 in the CNS and the spatiotemporal expression pattern from embryonic day 19.5 to adult brain using a recently described connexin45 LacZ-reporter mouse. The connexin45 gene is highly expressed during embryogenesis and up to 2 weeks after birth in nearly all brain regions. Afterward its expression is restricted to the thalamus, the CA3 region of hippocampus and the cerebellum. In adult mouse brain, the pattern of LacZ-staining in combination with the analysis of different neuronal and glial marker proteins strongly suggests that connexin45 is expressed in neurons, but presumably not in astrocytes or mature oligodendrocytes. Expression of the LacZ/connexin45 reporter gene in subsets of neurons, such as cerebral cortical, hippocampal and thalamic neurons as well as basket and stellate cells of cerebellum should be corroborated by functional investigations of connexin45 protein in electrical synapses. Based on its expression pattern during development, we suggest that the connexin45-containing gap junction channels have a rather ubiquitous role during brain development and may contribute to functional specification in certain subsets of neurons in the adult brain.